Peptide having an affinity for gp120

ABSTRACT

The peptide in this invention is a peptide having affinity to gp120 represented by Formula (1): H-A1-A2-A3-A4-A5-R  
     (in the formula,  
     H means hydrogen,  
     A1 is aspartic acid, lysine, valine, glutamic acid, glycine, asparagine, or tyrosine reidue,  
     A2 is valine, aspartic acid, tryptophan, lysine, phenylalanine, isoleucine, leucine, or tyrosine residue,  
     A3 is lysine, valine, aspartic acid, arginine, alanine, or tryptophan residue,  
     A4 is alanine, tryptophan, or glycine residue,  
     A5 is glycine, alanine, valine, leucine, isoleucine, serine, threonine, methionine, asparagine, glutamine, histidine, lysine, arginine, phenylalanine, tryptophan, proline, or tyrosine residue,  
     R is OH derived from carboxyl group or NH2 derived from acid amide group).  
     The above peptide has an affinity to gp120 of the HIV envelope protein and is superior in stability.

TECHNICAL FIELD

[0001] This invention relates to a peptide which has an affinity forgp120, HIV (human immunodeficiency virus) envelope protein.

BACKGROUND ART

[0002] The therapy for HIV infection is usually chemotherapy, such asthe nucleotide derivative AZT (3′-azido-3′-deoxythmidine). This AZTtherapy or protease inhibitor, which was later developed, prolongs thelife of HIV patients, but there are some problems, these are derivedfrom the chemotherapy itself.

[0003] There problems are shown as follows; The first is chronicpoisoning due to long term administration, the second is the appearanceof an HIV virus resistant to the medicine during the therapy, the thirdis the appearance of malignant tumors in prolongation of the HIVpatient's life, the fourth is that the recovery of the immune system cannot be monitored, the fifth is that the there not being a method tomonitor treatment effect, etc. Since such chemotherapy is not basictherapy for HIV infections, most people anticipate the development of avaccine.

[0004] Generally, the vaccine is an inactive treatment (in activevaccine) of a microbe of viruses; a weak activity virus which losespathogenesis or a pseudo virus (live vaccine) which has no fatal effectsto humans. However, although HIV itself is natively a weak activityvirus, it is well known to stay long after having once invaded the body.In addition, the host cell of HIV is mainly a lymphocyte, which controlsthe immune system; furthermore, HIV spreads over hemophilic patientsthrough blood-preparation. From these finding, even if it is assumedthat we selected either an in-active or a weak vaccine, the developmentof an HIV vaccine has many problems with safety.

[0005] Accordingly, an HIV vaccine is being developed which utilizes apart of the HIV envelope protein and inhibits further infection.

[0006] From such an idea, many researchers performed an epitope analysisof gp 120 in the HIV enveloped protein, and then, watched the V3 region(3rd hypervariable region) of gp120 as an epitope. But it was a truehypervariable region [Palker T. J., et al., Proc. Natl. Acad. Sci. USA85:2709-2713, 1988; Rusche J. R., et al., ibid 85:3198-3202,1988;Gouddsmit J., et all., ibid 85:4478-4482.1988; Matsushita S., et al., JVirol. 62:2107-2114,1988]. After this, a vaccine which used a part ofthis region as antigen was administrated to an HIV infected monkey as aninfection inhibitory experiment, but the effection has not yet beenreported.

[0007] As well as this, Tam et al. devised further antigenecity for theabove-mentioned peptide antigen (Tam et. al., Japanese patentpublication(Tokuhyo) No. H 3-503539), but have not yet had successbecause in most parts of the V region, particularly in the V3 which is aconvenient region for antibody preparation, mutation or deletion occurs.

[0008] In addition, a neutralized antibody, which inhibits the infectionagainst lymphocyte, is developed. For example, in Japanese PatentApplication No. 63-171385, after the production of a monoclonal antibodyby using a part of the above mentioned peptide as antigen, a method isreported, which produces anti HIV chimera antibodies on hybridizationdue to genetic engineering at the level of the protein as the Fab′itself. But, although with such neutralizing antibodies it is possibleto inhibit HIV infection to the lymphocyte at laboratory level, anantibody that can be used practically has not yet been developed.

[0009] As mentioned above, chemotherapy has some problems; drugtolerance in the virus and side effects in the host, another idea tosolve the problem of removing the virus from the body is byplasmapheresis. Although this method to remove the HIV virus by using apore size membrane filter (smaller than virus size) for plasmapheresishas been definitely proposed it is not yet possible to make a uniformpore size membrane. It is also possible that the pores will becomeclogged during plasmapheresis resulting in the deterioration of themembrane due to pressure. As mentioned above there are many technicalproblems which have to be settled. So, a method to use CD4 derived fromhuman lymphocyte having specific affinity to HIV, as absorbed carrier incolumn for plasmapheresis is also proposed. It cannot be used as amedical procedure because of the lost affinity due to decay by autoclavetreatment. In addition, there are also methods using thermostablemolecules, a high molecule polymer or color ligand as an affinitycarrier to HIV. However, as these molecules do not originally havespecific binding ability to HIV, they cannot be used because they bindto blood ingredients faster than to HIV.

[0010] In this way, aiming at the development of a HIV treatmentmedicine, research to produce a vaccine and neutrizing antibody isflourishing, but useful medicine has not yet been developed.

[0011] The inventors paid attention to this present situation anddeveloped a superior peptide to have the same degree or more affinityfor gp120 compared to antibodies and to be resistant to autoclavetreatment, and have already made a patent application (Japanese PatentApplication No. H 8-351474 and Japanese Patent Application No.8-351475). This peptide basically consists of a three amino acidsequence, but from a study of the sequel, we found that an affinity togp120 of this peptide deteriorated by number and a kind of the aminoacid which ranged in it. So, we knew that we needed to develop a morestable peptide.

DISCLOSURE OF THE INVENTION

[0012] In view of the above, an object of this invention is to provide anovel peptide which has an affinity for gp120, HIV envelope protein,with excellent stability, and a variety of usabilities using thepeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a graph that shows result of EXAMPLE 8.

[0014]FIG. 2 is a photograph by electron microscope that shows theresult of EXAMPLE 15.

BEST MODE FOR CARRYING OUT THE INVENTION

[0015] The No.1 peptide in this invention that could solve the abovesubject; A peptide having an affinity to gp120 represented by formula(1): H-A1-A2-A3-A4-A5-R

[0016] (in the formula,

[0017] H means hydrogen,

[0018] A1 is aspartic acid, lysine, valine , glutamic acid, glycine,aspragine, or tyrosine reidue,

[0019] A2 is valine, aspartic acid, tryptophan, lysine, phenylalanine,isoleucine, leucine, or tyrosine residue,

[0020] A3 is lysine, valine, aspartic acid, arginine, alanine, orTryptophan residue,

[0021] A4 is alanine, tryptophan, or glycine residue

[0022] A5 is glycine, alanine, valine, leucine, isoleucine, serine,threonine, methionine, asparagine, glutamine, histidine, lysine,arginine, phenylalanine, tryptophan, proline, or tyrosine residue, R isOH derived from carboxyl group or NH₂ derived from acid amide group).

[0023] Accordingly, the No. 1 peptide in this invention is a 5 aminoacid sequence that was constituted by A1, A2, A3, A4 and A5 as describedabove, and all of the peptide including such amino acid sequencescontained by the range of this invention. Thus, a peptide having anaffinity to gp120 represented by

[0024] Formula(2): A1′-A2-A3-A4-A5-R

[0025] (in the formula,

[0026] A1′ means aspartic acid, lysine, valine, glutamic acid, glycine,aspragine, or tyrosine reidue, or polypeptide residue that an arbitraryamino acid stood in line in the N-terminal side from this amino acid,A2, A3, A4, AS and R has the same meaning as above)

[0027] or

[0028] Formula (3): H-A1-A2-A3-A4-A5′-R

[0029] (in the formula,

[0030] A5′ means glycine, alanine, valine, leucine, isoleucine,

[0031] threonine, methionine, asparagine, glutamine, histidine, lysine,arginine, phenylalanine, tryptophan, proline, or tyrosine residue, orpolypeptide residue that an arbitrary amino acid stood in line in theC-terminal side of this amino acid, H, A1, A2, A3, A4 and R has the samemeaning as the above)

[0032] is entirely one aspect of the present invention.

[0033] Then, the No. 2 peptide that could solve the above subject is;

[0034] a peptide having an affinity to gp120 represented by

[0035] Formula(4): H-a1-a2-a3-a4-a5-R

[0036] (In the formula,

[0037] H means hydrogen,

[0038] a1 is tyrosine, arginine, phenylalanine, glycine, tryptophan,histidine, or asparatic acid reidue,

[0039] a2 is arginine, tyrosine, tryptophan, alanine, valine, glutamine,histidine, or lysine residue,

[0040] a3 is lysine, tyrosine, arginine, glutamic acid, methionine, ortryptophan residue,

[0041] a4 is glycine, alanine, valine, leucine, isoleucine, serine,threonine, methionine, asparagine, glutamine, histidine, lysine,arginine, phenylalanine, or tryptophan residue

[0042] a5 is glycine, alanine, valine, leucine, isoleucine, serine,threonine, methionine, asparagine, glutamine, histidine, lysine,arginine, phenylalanine, tyrosine, or tryptophan residue,

[0043] R is OH derived from carboxyl group or NH₂ derived from acidamide group).

[0044] Accordingly, the No.2 peptide in this invention is a 5 amino acidsequence that was constituted by a1, a2, a3, a4 and a5 as describedabove, and all of the peptide including such amino acid sequencescontained by the range of this invention. Thus,

[0045] a peptide having an affinity to gp120 represented by

[0046] Formula(5): a1′-a2-a3-a4-a5-R

[0047] (In the formula,

[0048] a1′ means tyrosine, arginine, phenylalanine, glycine, tryptophan,histidine, or asparatic acid reidue, or polypeptide residue that anarbitrary amino acid stood in the N-terminal side from this amino acid,a2, a3, a4, a5 and R has the same meaning as above.)

[0049] or

[0050] Formula(6): H-a1-a2-a3-a4-a5′

[0051] (In the formula, a5′ is glycine, alanine, valine, leucine,isoleucine, serine, threonine, methionine, asparagine, glutamine,histidine, lysine, arginine, phenylalanine, tyrosine, or tryptophanresidue, or polypeptide residue that an arbitrary amino acid stood inline in the C-terminal side of this amino acid, H, a1, a2, a3, and a4has the same meaning as above)

[0052] is entirely one aspect of the present invention.

[0053] In addition, this invention includes a compound which is amacromolecule compound that has a functional group and/or medicine boundto the No.1 or No.2 peptide as mentioned above, or a pharmaceuticallyacceptable salt thereof.

[0054] These peptide compounds or materials including them have anaffinity to gp120.

[0055] Furthermore, the above peptides or a pharmaceutically acceptablesalt thereof, and the composition including pharmaceutically acceptablecarrier and/or medicinal bioactivity, are contained in this invention.Also, various aspects, such as; the detection, the diagnosis and theremoval to viruses such as HIV by using the above peptide (for example,using the HIV diagnosis or the detection kit contained it, HIV absorbingand removal carrier, and therapy by plasmapheresis) are contained inthis invention.

[0056] However, the above mentioned “peptide” that is used in thisinvention contained in the C-terminal peptide is COOH, acid amide andester etc., and particularly, so long as we do not specify, it containsan aminino acid number (origopeptide) of less than 10, or a polypeptideof more than this.

[0057] An amino acid in the above mentioned peptide contains thederivatives that are protected by the protecting functional group. Assuch amino acid derivatives, it is marketed the substitution or themodification without exchanging the peptide structure; exchanging thelength of the carbon chain etc., or the protecting amino acidderivatives corresponding to various amino acids, but all of thesevarious amino acids can be used in this invention. For example, astyrosine derivatives, there is 2,6-dichloro-L-tyrosine having chloridein the side chain, p-Nitro-L-phenylalanine that hydroxyl group of p-sidein phenylalanine was substituted Nitroyl group, and4-chloro-L-phenylalanine that the hydroxyl group was substitutedchloride, etc. In addition, as valine derivaties, there are Norvaline:N-α-L-norvaline, or MeVal: N-α-L-valine, etc.

[0058] The reason that conventional medicine, such as a vaccine orneutrizing antibody can not be used clinically is that the HIV regionthe body can recognize as antigen is the V (hypervariable region) regionin the envelop gp120, and this is the most problematic. So, theinventors researched a peptide which had a specific affinity to gp120,and as a result, developed a superior peptide and have applied for apatent already (Japanese Patent Application No. H 8-351474 and JapanesePatent Application No. H 8-351475). They developed a peptide which has ahigh specific affinity to gp120, of the same affinity or more comparedto antibody , and which is additionally resistant to heat with a highpressure, such as in autoclave treatment.

[0059] However, from research after this, we found that theabove-mentioned affinity to gp120 deteriorated by number and there was akind of amino acid which ranged in it. So, we continued to researchfurther to supply a more stable peptide, and make this inventioncomplete.

[0060] In addition to the above, “affinity”, this invention shows aspecific and tight bond with weak interaction, such as electrostaticinteraction, hydrogen bond, Van der Waals attraction, hydrophobic bondand etc, gathered except a covalent bond.

[0061] The peptide in this invention is constituted as mentioned aboveand is fundamentally shown as 5 amino acid residues which appeared in;

[0062] {circle over (1)}formula (1): H-A1-A2-A3-A4-A5-R (in formula, A1,A2, A3, A4, A5 and R the meaning is the same as before) or,

[0063] {circle over (2)}formula (4): H-a1-a2-a3-a4-a5-R (in formula, a1,a2, a3, a4, a5 and R the meaning is the same as before)

[0064] This peptide has a molecule separate in each and is not(or inpeptide), the amino acid sequence mentioned above {circle over(1)}peptide,

[0065] formula (2): A1′-A2-A3-A4-A5; or

[0066] formula (3): A1-A2-A3-A4-A5′

[0067] or above mentioned {circle over (2)}peptide includes the sequencewhich lined up from N-terminus.

[0068] Formula (4): a1′-a2-a3-a4-a5; or

[0069] Formula (5): a1-a2-a3-a4-a5′

[0070] (in formula, A1′, A2, A3, A4, A5′, a1′, a2, a3, a4, a5′ themeaning is the same as before)

[0071] Of course, in, A1′-A2′-A3′-A4′-A5′ or a1′-a2′-a3′-a4′-a5′ thereincludes peptides which lined up repeatedly by this order. In brief,this invention includes all of the peptides which consist of 5 aminoacid residues and have an affinity to gp120.

[0072] The peptide in this invention can be synthesized by conventionalmethods; For example, the first of this invention is constituted fromA1-A2-A3-A4-A5, is synthesized and the A5 glycine residue, carboxyl ofN-protective glycine is bound to some carrier, such as insoluble resin,which has a functional group that can couple to carboxyl. After this,the protected amino acid in each, from A2 to A5, is bound in order by asolid phase synthetic method, and the peptide shown in this inventioncan be got by reacting the above mentioned insoluble resin andeliminating the protection of the amino acid.

[0073] In addition to the above, an end of carboxyl in A5 amino acidresidue is free(R, that is to say is equivalent to —OH), or issubstituted with acidic amide(R, that is to say is equivalent to —NH₂).Then, an end-carboxyl of A5 with carboxyl of spacer together, bound thiscarboxyl, binds a synthetic macromolecule, bio-macromolecule, andutilized well macromolecular compound, which has a functional group (asin the postscript). In addition to the above, amino acid used by theabove mentioned solid phase synthetic methods is common to L type or Dtype, but L type is more pleasing.

[0074] In the case of the above, the carrier used solid phase syntheticmethod has carboxyl group of N-protected glycine of C-terminus throughthe amino group, or if it can bind to this carboxyl group and caneliminate after the binding, it is not limited at all. For example,chloromethyl-resin (chloromethylpolystyrenedivinylbenzene),oxymethyl-resin (oxymethylpolystyrenedivinylbenzene) and others areexemplified. Then, resin of 4-(oxymethyl)phenylacetamidemethyl-resin,4-(2′,4′-Dimethoxyphenyl-aminomethyl)phenoxyacetamidemethyl-resin andetc., benzyloxybenzylalcohol-resin, benzhydrylamine-resin,insoluble-resin which has amino group, methylbenzhydrylamine-resin,aminomethylphenoxymethyl-resin, dimethoxybenzhydrylamine (DMBHA)-resin,and the derivatives are exemplified. In these, benzhydrylamine-resin,methylbenzhydrylamine-resin, aminomethylphenoxymethyl-resin andDMBHA-resin can be get directly amide by cleavage after the binding.Judging from the yield, use of aminomethyl-resin is desirable.

[0075] In addition, a spacer which has a functional group binding withcarboxyl group and has a carboxyl group are picked up which cantransform p-carboxymethylbenzylester-resin to carboxyl group of glycineas in the example.

[0076] Moreover, “protecting amino acid” in case of above is meaningprotected amino acid with protecting group by conventional method. Tosynthesize the peptide invented, it is pleasing to use either of theprotecting groups shown in the following examples.

[0077] In this example, the protecting group of α-amino in amino acid isBoc (t-butoxycarbonyl) or Fmoc (9-fluorenylmethoxycarbonyl); protectinggroup of ξ-amino in lysine is Z (benzyloxycarbonyl),Cl.Z(2-chlorobenzyloxycarbonyl), Boc, Npys(3-nitro-2-pyridinesulfonyl);protecting group of hydroxyl group in tyrosine is Bzl(benzyl),Cl₂.Bzl(2,6-dichlorobenzyle), or t-Bu(tert.-butyl) are exemplified, butit uses the peptide synthesis well even if the hydroxyl group of thistyrosine is not protected by the above mentioned protecting group;protecting group of guanidino group in arginine is Tos(tosyl),NO₂(Nitro), Mtr(4-methoxy-2,3,6-trimethylbenzenesulfonyl), orpmc(2,2,5,7,8-pentometylchloroman-6-sulfonyl); protecting group ofcarboxyl group in glutamic acid is Bzl ester, t-Bu ester, cHx(cyclohexlylester); protecting group of amide group in glutamine isTrt(trityl) is picked up, but it can be used even if glutamine is notprotected by this protecting group; the protecting group of indole groupin tryptophan is formyl group or Boc, but it can be used even iftryptophan is not protected by this protecting group. These protectinggroups can be used to select the most suitable protecting amino acidaccording to the condition of the peptide synthesis.

[0078] A binding of protecting amino acid can be carried out with theusual condensed-polymerized method, as for example, DCC(N′,N-dicyclohexylcarbodiimide)method(R. B. Merrified: Biochemistry, 3,1385, 1964), DICDI(N′,N-diisopropylcarbodiimide) method(D. Sarantakis,et al: Biochem. Biophys. Res. Commun., 73,336,1976), active-estermethod(F. Weygan, et al.: Z. Naturforsch., B, 21, 1141,1966), mixturedor symmetrical acetic anhydride method(D. Yamashiro, et al.: Proc. Natl.Acad. Sci. USA, 71, 4945,1945) carbonyldiimidazole method,DCC-HOBt(1-hydroxybenzotriazole) method(Keonig, W., et al.; Chem. Ber.,103: 788,1970), diphenylphosphorylazide method, etc. But particularly,DCC, DCC-HOBt, DICDI-HOBt and symmetric acidic anhydride method arerecommended. These condensed—polymerized reactions are usually performedin organic solvent of dichloromethane, dimethylformamide etc. or intheir mixture solution.

[0079] As in the case above, reagents to eliminate the protecting groupof α-amino group, trifluoroaceticacid(TFA)/dichloromethane, HCl/dioxane,piperidine/dimethylformamide(DMF), etc. are used and can be selectfreely according to the kind of protecting group. Then, the degree ofprogress in the condensed reaction on each step of synthesis can beconfirmed by the ninhydrin reaction method (E.Kaiser, et al., Anal.Biochem., 34:595,1970).

[0080] In this way, we can get protected peptide resin which has anamino acid sequence represented with an upper expression, and afterthis, can get a suitable peptide by elimination of the protecting groupfrom the insoluble resin and amino acid. In the case using chloromethylresin as insoluble resin, it is treated by hydrogen fluoride withanisole. Then, in this case used benzyloxybenzylalcohol-resine,benzhydrylamine-resin, and DMBHA resin(Funakoshi, S., J. Chem. Soc.,Chem. Commun., 198: 382,1988), this resin and protecting group can beeliminated at same time by treatment of hydrofluore, TFMSA(Trifluoromethanesulfonicacid), TMSOTF (Trimethylsilyltriflate), TMSBr(Trimethylsilylbromide) and others.

[0081] The peptide that is got in this way can be purified by variousmethods; chromatography (gel, ion-exchange, hydrophobicity, adsorption,reverse phase), electrophoresis and ultrafiltration.

[0082] Also included in this invention is a peptide that was substitutedon a similar protein(active center or binding domain of antibody,receptor, enzyme and etc.) by a gene recombination method in the case ofabove peptide. For example, if we produce human anti-gp120 antibody bygene recombinant method, we produce the above-mentioned peptide which isbased on the U.S. Pat. No. 114632. Namely, this peptide is transducedamino acids of hypervariable cluster in CDR (coplementaritydetermination region, VH31 to VH35)-1 and CDR-2(VH50 to 52, and/or VH58to 60), which relates recognition of epitope during the V region in thehuman immunoglobin gene (Ohno, S., Mori, N. & Matunaga, T.; Proc. Natl.Acad. Sci. USA. 82, 2945, 1985).

[0083] In this way, the peptide of this invention can produce specificbinding to, gp120 accordingly to its purpose, substituting the generecombinant method.

[0084] For example, the first peptide in this invention is shown fromtable 1 to 2 and at same the second peptide is shown from table 3 to 4,respectively. As in the case above, the table shows an effectiveagglutinin test and neutralizing activity. Here, No. 24 in Table 1 isequivalent to the first peptide, and also matches the second peptide.TABLE 1 Agglutinin Neutralizing No. A1 A2 A3 A4 A5 test activity 1 AspVal Lys Ala Gly * 2 Asp Lys Val Ala Gly * 3 Lys Val Asp Ala Gly * 4 ValLys Lys Ala Gly * 5 Asp Asp Lys Ala Gly * 6 Lys Asp Asp Ala Gly * 7 ValAsp Asp Ala Gly * 8 Asp Val Asp Ala Gly * 9 Val Val Lys Ala Gly * * 10Val Val Asp Ala Gly * 11 Lys Val Val Ala Gly * 12 Asp Asp Val AlaGly * * 13 Asp Asp Asp Ala Gly * 14 Val Lys Val Ala Gly * 15 Asp Try LysAla Ala * 16 Asp Phe Lys Ala Gly * 17 Asp Trp Lys Ala Gly * 18 Asp ValArg Ala Gly * 19 Glu Val Lys Ala Gly * 20 Gly Gly Asp Val Lys Ala Gly *21 Gly Asp Val Lys Ala Gly * 22 Val Ile Asp Ala Gly * 23 Val Leu Asp AlaGly * 24 Gly Val Lys Ala Gly * 25 Asp Val Lys Trp Ala * 26 Asp Val LysGly Lys * 27 Asp Val Lys Gly Trp *

[0085] TABLE 2 agglutinin neutralizing No. A1 A2 A3 A4 A5 test activity28 Asp Val Ala Ala Gly * 29 Asp Val Lys Gly Leu * 30 Asp Val Lys GlyPro * 31 Asp Val Lys Ala Val * 32 Asp Val Lys Ala Ile * 33 Asp Val LysAla Ser * 34 Asp Val Lys Ala Thr * 35 Asp Val Lys Ala Met * 36 Asp ValLys Ala Gln * 37 Asp Val Lys Ala Asn * 38 Asp Val Lys Ala His * 39 AspVal Lys Ala Arg * 40 Asp Val Lys Ala Phe *

[0086] TABLE 3 Agglutinin Neutralizing No. A1 A2 A3 A4 A5 test activity1 Phe Tyr Arg Lys Ala * * 2 Tyr Arg Arg Ala Ala * 3 Trp Trp Glu AlaAla * * 4 Tyr Gln Glu Ala Ala * 5 Gly Tyr Tyr Lys Ala * * 6 Trp Trp LysAla Ala * * 7 Tyr Tyr Arg Ala Ala * 8 Phe Arg Lys Ala Ala * 9 Tyr TyrLys Lys Ala * * 10 Tyr Tyr Lys Leu Leu * 11 Tyr Arg Lys Ala Ala * * 12Tyr Tyr Lys Ala Ala * * 13 Arg Tyr Lys Ala Ala * * 14 Phe Tyr Arg AlaAla * 15 Tyr Ala Lys Ala Ala * * 16 Tyr Tyr Glu Ala Ala * 17 Tyr Trp LysAla Ala * 18 Gly Tyr Tyr Lys Ala Ala * 19 Typ Tyr Lys Ala Ala * 20 TyrGln Lys Ala Ala * 21 His Tyr Lys Ala Ala * 22 Tyr Arg Tyr Ala Ala * * 23Tyr Tyr Met Ala Ala * 24 Tyr Val Lys Ala Ala * 25 Gly Tyr Ala Tyr ArgLys * 26 Arg Arg Trp Ala Tyr * * 27 Arg Tyr Tyr Lys Ala Ala *

[0087] TABLE 4 Agglutinin Neutralizing No. A1 A2 A3 A4 A5 test activity28 Tyr Lys Lys Ala Ala * 29 Tyr His Lys Ala Ala * * 30 Asp Tyr Lys AlaAla * 31 Tyr Tyr Lys Trp Ala * 32 Tyr Tyr Lys Gly Ala * 33 Tyr Tyr LysAla Gly * 34 Tyr Tyr Lys Lys Ala * 35 Tyr Tyr Lys Val Ala * 36 Tyr TyrLys Ile Ala * 37 Tyr Tyr Lys Ser Ala * 38 Tyr Tyr Lys Thr Ala * 39 TyrTyr Lys Met Ala * 40 Tyr Tyr Lys Gln Ala * 41 Tyr Tyr Lys Asn Ala * 42Tyr Tyr Lys His Ala * 43 Tyr Tyr Lys Phe Ala * 44 Tyr Tyr Lys Trp Ala *45 Tyr Tyr Lys Arg Ala * 46 Tyr Tyr Lys Ala Val * 47 Tyr Tyr Lys AlaIle * 48 Tyr Tyr Lys Ala Ser * 49 Tyr Tyr Lys Ala Thr * 50 Tyr Tyr LysAla Met * 51 Tyr Tyr Lys Ala Gln * 52 Tyr Tyr Lys Ala Asn * 53 Tyr TyrLys Ala His * 54 Tyr Tyr Lys Ala Phe * 55 Tyr Tyr Lys Ala Trp * 56 TyrTyr Lys Ala Arg *

[0088] A sign of each amino acid formula shows the amino acid residue bythe internationally approved characters, the details are as follows:

[0089] Tyr: Tyrosine

[0090] Lys: Lysine

[0091] Trp: Tryptophan

[0092] Arg: Arginine

[0093] Glu: Glutamic acid

[0094] Gln: Glutamine

[0095] His: Histidine

[0096] Ala: Alanine

[0097] Phe: Phenylalanin

[0098] Gly: Glycine

[0099] Met: Methionine

[0100] Asp: Asparatic acid

[0101] Asn: Asparagine

[0102] Val: Valine

[0103] Ser: Serine

[0104] Cys Cystine

[0105] Thr: Threonine

[0106] Ile: Isoleucine

[0107] Leu: leucine

[0108] Pro: Proline

[0109] A peptide having such an amino acid sequence shows a superioraffinity to gp120, and can be utilized effectively as an anti-HIVmedicine by taking a form of chemical compound or composition shown asfollows.

[0110] A compound of this invention is matter that binds a highmolecular chemical compound and/or medicinal activator functional group,and this invention includes the salts to be admitted as medicine.

[0111] For example, as pharmaceutically acceptable salts here, followingintoxicant salts in common use is put up.

[0112] {circle over (1)}As salts with bases such as inorganic bases,there are alkali metal salt (for example, sodium salt and potassiumsalt), alkaline earth metal salt (for example, calcium salt, magnesiumsalt) and ammonium salt; {circle over (2)} as salts with such as organicbases, there is salts of organic amines (for example, triethylamine,pyridine, picoline, ethanolamine, triethanolamine, dicyclohexylamine,N,N′-dibenzylethylenediamines), etc; {circle over (3)} as salts withacids such as inorganic acids, there are hydrocholic acid, hydrobromicacid, phosphoric acid, nitric acid, sulfuric acid; {circle over (4)} assalts with acids such as organic acids, there are organic carboxylicacid (for example, acetic acid, propionic acid, maleic acid, succinicacid, malic acid, citric acid, tartaric acid and salicylic acid),organic sulfonic acid (for example, methanesulfonic acid,p-toluenesulfonic acid ), glyconic acid (glucuronic acid, galactonicacid, gluconic acid, ascorbic acid , and others).

[0113] Then, “macromolecule compound having functional group” used onthis invention is not particularly limited if it can bind the peptide ofthis invention, for example, the following are listed.

[0114] (1) Synthetic Polymer

[0115] It is selected voluntarily among the inside of linear, branchedand cyclic, as polymer in case of above. For example, it can be used asan insoluble solid phase carrier of amino acidic homopolymer ofpolylysine and polyglutamic acids, or cyclic polyaimne, cyclodextrin,cyclic peptide and then polystyrene, polypropyrene, nylon, silica-gel,polyethyleneglycol, cellulose, polyacrylamide, and others.

[0116] A branched polymer in these is higher than the usual homopolymeron content of functional groups per unit because of divergence at onepart in each. For example, it seems to be the lysine core indicated byDenkewalter and is a polymer that is based on the same molecular chain,more than two, derived from a core molecule having at least two or morehaving functional groups U.S. Pat. No. 4,289,872; or it is a starburstdendrimer that the polymer size is regulated closely because the samemolecule reacts continuously, proposed by Tomalia and et al; or it isthe molecule that size was formed irregular , by which the same ordifferent molecule reacts to discontinuity. In addition, ahomo-/branched-polymer as in the case of above does not always to need acarrier which has enough size, and it has a monomer of around 3 thatdoes not usually seem to be recognized as a core, and it is not limitedby the size or introduction number. However, in the case when it isintroduced to numerous peptide formulas, use of divergence numericalpolymer is recommended but even if it is anything polymer. When thepeptide of this invention is bound (to the above mentioned polymer) itis possible that it is synthesized and just grown directly/indirectlyfrom the branched functional group, or that it is conjugateddirectly/indirectly from the functional group of the polymer to a newseparate synthetic peptide.

[0117] Moreover, for binding cyclic polymer of cyclic polyamine,cyclodextrin, and cyclic peptides, it is possible to synthesis directlyand make the peptide of upper expression from the same functional group,or to bind directly/indirectly to a new synthetic peptide separately orto a functional group of the cyclic polymer. Then, to bind the insolublecarrier of silica gel etc., after it is introduced to the samefunctional group carrier in advance, it can be synthesized and just growdirectly peptide of upper expression from the functional group, orconjugate directly/indirectly from the functional group of insolublecarrier to the new synthetic peptide separately. In addition, theparticular size and shape of the carrier having this same functionalgroup is not limited, and selection and utilization of: spherical,hollow fibrous, fibrous shapes can be made according to their purposeand then, it is not limited at all by size and shape and can beintroduced to several functional groups.

[0118] (2) Biopolymer

[0119] As above mentioned biopolymer, there are, for example, linearpolymer like polysaccharide, such as heparin, hyaluronic acid, chitosan,chitin, etc.; proteins of proteoglycans, peptide hormone; gelatin,albumin, antibody, and antibody's fragments, etc.

[0120] The size of linear polymer in these can be an appropriatelyselected, according to the purpose of use, and includes some monomer ofaround 3 that does not usually seems to be recognized as a polymer, butis not limited at all by the size or number of functional groups. Forbinding a peptide of upper expression to this linear polymer, you maydirectly synthesis and just grow it from the same functional group, ormay directly/indirectly conjugate a new synthetic peptide separately tothe functional group of the linear polymer.

[0121] In addition, when peptide hormone and protein is bound to thepolymer, it is possible that either end of the peptide of upperexpression is bound to cysteine and do disulfide binding with theresidue of cysteine in the above mentioned peptide hormone/protein, oris conjugated directly/indirectly to the functional group in the abovementioned peptide hormone/protein with functional group peptide in upperexpression. In this way, we can freely select these binding methodsaccording to the purpose of use, and also it is the same even if thekind and number of peptide shown by upper expression is transduced.

[0122] Moreover, an active medicine used by this invention is, forexample, AZT of nucleoside derivative known as HIV inhibitor,3,4-Duhydroxy-2,5-di[N-methyl-(2-pyridylmethyl)carbamoyl]valylamino]-1,6-diphenylhexanes known as protease inhibitoragainst HIV. These active medicines can be used to produce specificmedicines against HIV. Accordingly, such a medicine is useful as thetreatment medicine that can specifically treat HIV.

[0123] Furthermore, the range of this invention also includes acomposition that contains a salt and a carrier pharmaceuticallypermitted as medicine and/or active medicine.

[0124] As mentioned above [the pharmaceutically permitted carrier] canselect the appropriate use of an excipient (disintegrator, lubricant,expander etc.), color additives, preservative, stabilizer and othercarriers in common use. The following have definitely been shown crystalcellulose, calcium carmelose, sodium carmelose, hydroxypropylcellulose,hydroxypropylmethylcellulose, ethylcellulose, magnesium stearate, talc,light anhydrouse silicic acid, food color agents, essential oil, etc.

[0125] Based on the following enforcement examples, we will describe thedetails of this invention. However, the following enforcement exampledoes not limit this invention, the technological range of this inventionincludes all enforcing changes within the range that does not deviatefrom the purpose of the postscript.

Synthesis 1: Compound which was Bound Polyethylene Glycol with thePeptide in this Invention

[0126] After induction of carboxyl group by which was reacted anhydridesuccinic acid against a hydroxyl group of polyethylene glycol (MW.20,000), it is reacted MBS (m-maleimide benzoyl-N-hydroxysuccinimide),and we synthesized maleimide polyethylene glycol.

[0127] Then, we synthesized peptide-polyethylene glycol bindingcompound, by which performed peptide binding to peptide induced cysteineagainst C-terminal of No.1 peptide shown by Table 1 mentioned above.After suspending this compound in phosphate buffer solution, it waspurified by gel chromatography and affinity chromatography by gp120conjugated carrier, and then, polyethylene glycol binding compound tothe peptide was synthesized.

Synthesis 2: Compound which was Bound Cyclodextrin with the Peptide inthis Invention

[0128] After induced carboxyl group by which was reacted anhydridesuccinic acid against a hydroxyl group of α-cyclodextrin, it was reactedMBS(m-maleimide benzoyl-N-hydroxysuccineimide), and we synthesizedmaleimide cyclodextrin.

[0129] On the other hand, after synthesized a peptide which wasperformed peptide binding to C-terminus of cysteine of No.12 peptide onTable 3 mentioned above, it reacted to the above maleimide cyclodextrin,and we synthesized a cyclic compound that cyclodextrin binding compoundto the peptide.

Synthesis 3: Compound (1) which was Bound Branched Polymer with thePeptide in this Invention

[0130] Branched polymer binding compound was synthesized by extendingNo.1 peptide on the above Table 1 from N-end amino acid MAPs (Multipleantigenic peptide). After the compound was suspended in phosphatebuffer, it was purified by gel chromatography and affinitychromatography by gp120 conjugated carrier, and then, we synthesizedbranched polymer binding compound to the peptide in this invention (1).

Synthesis 4: Compound (2) which was Bound Branched Polymer with thePeptide in this Invention

[0131] Branched polymer binding compound was synthesized by extendingNo.12 peptide on the above Table 3 from N-end amino acid MAPs (Multipleantigenic peptide). After the compound was suspended in phosphatebuffer, it was purified by through gel chromatography and affinitychromatography by gp120 conjugated carrier, and we synthesized branchedpolymer binding compound to the peptide in this invention (2).

Synthesis 5: Compound which was Bound AZT with the Peptide in thisInvention

[0132] After preparing mixed anhydride to react isobutyl chloroformatewith bromoacetic acid, we synthesized bromoacetylester-AZT byesterification, of the hydroxyl group of AZT. On the other hand, aftersynthesizing by solid phase method binding the peptide that cysteine wasbound C-end amino acid of No. 12 peptide on the above Table 3, it wasreacted with the above bromoacethylestel-AZT, and then we obtained AZTbinding compound, which is a cross-linking with this peptide.

Synthesis 6: Compound which was Bound Inactive Alkaline Phosphatase withthe Peptide in this Invention

[0133] We synthesized maleimide alkaline phosphatase, by which inactivealkaline phosphatase (Alp) was reacted MBS (maleimidebenzoile-N-hydroxysuccinimide). Then, No.8 peptide on the above Table 3induced cysteine was bound with the maleimide group, and we synthesizedinactive alkaline phosphatase binding compound. An inactive form ofalkaline phosphatase was identified by the confirmation that there wasno production of p-nitrophenol when using p-nitrylphenyl phosphate as asubstrate.

Synthesis 7: An Absorbing and Removing Carrier (1) which was BoundSephadex 6MB with the Peptide in this Invention

[0134] No.1 peptide on the above Table 1 was bonded covalently toSephadex 6MB by intermediating an activated spacer beforehand, andpeptide/Sepharose 6MB(1 μmol of gp120/ml in bed volume) was prepared.Unreactive peptide was centrifuged at room temperature for 10minutes(12,000 rpm) by using phosphate buffer and was removed byrepeating the absorbing and removing the preparation to the supernatant.

Synthesis 8: An Absorbing and Removing Carrier (2) which was BoundSephadex 6MB with the Peptide in this Invention

[0135] No.12 peptide on the above Table 3 was done covalent bond toSephadex 6MB by intermediating an activated spacer beforehand, andpeptide/Sepharose 6MB(1 μmol of gp120/ml in bed volume) was prepared.Unreactive peptide was centrifuged at room temperature for 10minutes(12,000 rpm) by using phosphate buffer and was removed byrepeating the absorbing and removing the preparation to the supernatant.

Synthesis 9: an Absorbing and Removing Carrier which was Bound Sephadex4B with the Peptide in this Invention

[0136] No.12 peptide on the above Table 3 was done covalent bond toSephadex 4B by intermediating an activated spacer beforehand, and thepeptide/Sepharose 4B(1 μmol of gp120/ml in bed volume) was prepared.Unreactive peptide was centrifuged at room temperature for 10minutes(12,000 rpm) by using phosphate buffer and was removed byrepeating the absorbing and removing the preparation to the supernatant.

Synthesis 10: an Absorbing and Removing Carrier which was BoundCellulose Type Carrier with the Peptide in this Invention

[0137] No.12 peptide on the above Table 3 was done covalent bond to aperoxycellulose carrier, and it was prepared by washing carefully withsodium hydrogen carbonate buffer(pH8) and phosphate buffer(pH4) afterblocking by glycine. We prepared an absorbing and removing carrier whichwas bound cellulose series carrier with the peptide.

EXAMPLE 1 Investigation of Neutralizing Activity

[0138] In this example, we investigated neutralizing activity of HIV-1using various peptides, shown in Tables 5 and 6.

[0139] We added separately 50 μL of the above peptide; 50 μL of200TCID⁵⁰ of HTLV-IIIB (laboratory strain) and KK-1(freshly isolatedHIV, Ohtake et al. Kansenshou(Japanese), 64,1284-1294,1990) in each; and50 μL of the above peptide precisely diluted by 2 times step-by-step in96-well microplate, and mixed. Then, we used AZT as a positive control.

[0140] After incubation for 30 minutes at 37° C., 100 μL of MT-4 cellsuspension of 3×10⁴ cells was added and incubated for 6 days at 37° C.under 98% of humidity and 5% of CO₂. After the incubation, cellplasmodium effect (CPE) by HIV-1 prolification, namely when a medicinewas added with dilution step-by-step and infection cells gathered andbecame a state to form an island(focus form), was judged as an intensityof neutralizing activity(content to inhibit infection) on a pre-stage ofthis diluted magnification. These results are shown jointly in Tables 5and 6. TABLE 5 HIV neutralizing activity Amino acid sequence (μg/ml) No.A1 A2 A3 A4 A5 HThV-IIIB KK-1 1 Val Lys Lys Ala Gly Nglu 15.6 2 Asp AspLys Ala Gly 62.5 NE 3 Val Val Lys Ala Gly 31.3 NE 4 Aps Asp Val Ala Gly500 NE 5 Asp Asp Asp Ala Gly 1,000 1,000 6 Val Lys Val Ala Gly 125 NE 7Asp Tyr Lys Ala Ala 31.3 125

[0141] TABLE 6 Anti-HLV neutralizing activity Amino acid sequence(μg/ml) No. a1 a2 a3 a4 a5 HTLV-IIIB KK-1 1 Phe Tyr Arg Lys Ala 250 1252 Tyr Arg Arg Ala Ala 250 125 3 Trp Trp Glu Ala Ala 250 NE 4 Gly Tyr TyrLys Ala 31.3 125 5 Tyr Tyr Ala Ala Ala 250 250 6 Phe Arg Lys Ala Ala 125125 7 Tyr Tyr Lys Lys Ala 312.5 312.5 8 Tyr Tyr Lys Leu Leu 31.3 62.5 9Tyr Arg Lys Ala Ala 312.5 156.3 10 Tyr Tyr Lys Ala Ala 78.1 39.1 11 ArgTyr Lys Ala Ala 62.5 31.25 12 Phe Tyr Ala Ala Ala NE 250-125 13 Tyr AlaLys Ala Ala 500 NE 14 His Tyr Lys Ala Ala NE 500 15 Tyr Arg Met Ala Ala31.25 31.25 16 Tyr Tyr Met Ala Ala NE 125 17 Tyr Val Lys Ala Ala NE 25018 Arg Arg Trp Ala Tyr 39.1 39.1 19 Tyr His Lys Ala Ala 500 500

[0142] As is clear from the result of Tables 5 and 6, neither peptidewhich was not satisfied with a matter of this invention showedneutralization activity to HIV-1, while the peptides which satisfied itshowed superior activity. Then, a peptide of this invention shown inTable 5 presents No.1 peptide of this invention, and the peptide ofTable 6 shows the No.2, in each. In addition to the laboratory strain,when this invented peptide showed superior activity to the freshlyisolated strain, this suggested it would be extremely useful at apractical level beyond the laboratory.

EXAMPLE 2 Agglutinin Test

[0143] In this example, we used the peptide shown in Table7-10, andevaluated it by an agglutinin test for the affinity to gp120.

[0144] After mixing equally both a suspension of 1% activation latexbeads (Polyscience Inc., particle size is 0.2 mm) and avidin(10 mg/mL).We incubated for 1 hour at 37° C. After the incubation, we added bovineserum albumin(BSA, 1 mg/mL) in it and carried out blocking of thereactive site for 30 minutes at 37° C. Then, after removing the bindingreagent by centrifuging repeatedly, biotinylated peptide (10 mg/mL inphosphate buffer, pH7.5) was added to each, and anti-gp120 agglutininreagent was prepared by incubation for 1 hour at 37° C.

[0145] As a positive control, we used colloidal gold conjugated byrecombinant gp120 that was expressed in baculo virus(ImmunodiagnosticsInc., particle size is 30 nm), while as a negative control, we used thisrecombinant gp120 that was dissolved in phosphate buffer.

[0146] 20μ of the above agglutinin reagent and positive control wereadded and mixed on a test disk separately, we investigated it with thenaked eye after standing for 10 minutes. The results are shown jointlyin Tables 7 to 10. When we used a negative control instead of a positivecontrol in this enforcement example, we confirmed that the agglutinationwas not observed. TABLE 7 Amino acid sequence Agglutinin test No. A1 A2A3 A4 A5 gp120/C.G. 1 Asp Val Lys Ala Gly +++ 2 Asp Lys Val Ala Gly + 3Lys Val Asp Ala Gly +++ 4 Asp Asp Lys Ala Gly + 5 Lys Asp Asp Ala Gly ++6 Val Asp Asp Ala Gly ++ 7 Asp Val Asp Ala Gly + 8 Val Val Lys Ala Gly +9 Val Val Asp Ala Gly 10 Lys Val Val Ala Gly + 11 Asp Tyr Lys Ala Ala +12 Asp Phe Lys Ala Gly ++ 13 Asp Trp Lys Ala Gly ++ 14 Asp Val Ar AlaGly + 15 Glu Val Lys Ala Gly ++ 16 Gly Gly Asp Val Lys Ala Gly 17 GlyAsp Val Lys Ala Gly ++ 18 Val Ile Asp Ala Gly + 19 Val Leu Asp Ala Gly +20 Gly Val Lys Ala Gly + 21 Asn Val Lys Ala Gly +++ 22 Asp Val Lys TrpAla + 23 Asp Val Lys Gly Lys + 24 Asp Val Lys Gly Trp + 25 Asp Val LysGly Leu + 26 Asp Val Lys Gly Pro +

[0147] TABLE 8 Amino acid sequence Agglutinin test No. A1 A2 A3 A4 A5gp120/C.G. 27 Asp Val Lys Ala Val + 28 Asp Val Lys Ala Ile + 29 Asp ValLys Ala Ser + 30 Asp Val Lys Ala Thr + 31 Asp Val Lys Ala Met + 32 AspVal Lys Ala Gln + 33 Asp Val Lys Ala Asn + 34 Asp Val Lys Ala His + 35Asp Val Lys Ala Arg + 36 Asp Val Lys Ala Phe +

[0148] TABLE 9 Amino acid sequence Agglutinin test No. a1 a2 a3 a4 a5gp120/C.G. 1 Phe Tyr Arg Lys Ala + 2 Trp Trp Glu Ala Ala + 3 Tyr Gln GluAla Ala + 4 Gly Tyr Tyr Lys Ala + 5 Trp Trp Lys Ala Ala +++ 6 Tyr TyrLys Lys Ala + 7 Tyr Arg Lys Ala Ala + 8 Tyr Tyr Lys Ala Ala + 9 Arg TyrLys Ala Ala + 10 Tyr Ala Lys Ala Ala + 11 Tyr Tyr Gln Ala Ala + 12 TyrTrp Lys Ala Ala + 13 Gly Tyr Tyr Lys Ala Ala + 14 Trp Tyr Lys Ala Ala +15 Tyr Gln Lys Ala Ala ++ 16 His Tyr Lys Ala Ala + 17 Tyr Arg Tyr AlaAla ++ 18 Gly Tyr Ala Tyr Arg Lys + 19 Arg Arg Trp Ala Tyr + 20 Arg TyrTyr Lys Lys Ala Ala + 21 Tyr Lys Lys Ala Ala + 22 Tyr His Lys Ala Ala +23 Asp Tyr Lys Ala Ala + 24 Tyr Tyr Lys Trp Ala + 25 Tyr Tyr Lys GlyAla + 26 Tyr Tyr Lys Ala Gly +

[0149] TABLE 10 Amino acid sequence Aggiutinin test No. a1 a2 A3 a4 a5gp120/C.G. 27 Tyr Tyr Lys Lys Ala + 28 Tyr Tyr Lys Val Ala + 29 Tyr TyrLys Ile Ala + 30 Tyr Tyr Lys Ser Ala + 31 Thr Tyr Lys Thr Ala + 32 TyrTyr Lys Met Ala + 33 Tyr Tyr Lys Gln Ala + 34 Tyr Tyr Lys Asn Ala + 35Tyr Tyr Lys His Ala + 36 Tyr Tyr Lys Phe Ala + 37 Tyr Tyr Lys Trp Ala +38 Tyr Tyr Lys Arg Ala + 39 Tyr Tyr Lys Ala Val + 40 Tyr Tyr Lys AlaIle + 41 Tyr Tyr Lys Ala Ser + 42 Tyr Tyr Lys Ala Thr + 43 Tyr Tyr LysAla Met + 44 Tyr Tyr Lys Ala Gln + 45 Tyr Tyr Lys Ala Asn + 46 Tyr TyrLys Ala His + 47 Tyr Tyr Lys Ala Phe + 48 Tyr Tyr Lys Ala Trp + 49 TyrTyr Lys Ala Arg +

[0150] From the results in Tables 7-10, we can confirm that each peptideshown in this invention has a superior affinity to gp120. The peptide ofthis invention is shown in Tables 7 and 8 is the No.1 peptide in thisinvention, and it shown in Tables 9 and 10 is the No.2 peptide of it.

EXAMPLE 3 Effect of A4 and A5 in this Invention on the Agglutinin Test

[0151] To examine the effect of A4 and A5 amino acid on the aggluitinintest, the chain length of the No.1 peptide in TABLE 7 was changed. Theagglutinination was measured the same as in 2. These results are shownjointly in TABLE 11. Face note, ± means “agglutinination in tracedegree”. TABLE 11 Amino acid sequence Agglutinin test No. A1 A2 A3 A4 A5gp120/C.G. 1 Asp Val Lys Ala Gly +++ 2 Asp Val Lys Ala — + 3 Asp Val Lys— — ±

[0152] From TABLE 11, both No.3 with only 3 amino acids (there are no a4and a5 amino acids) and in No.2 with only 4 amino acids(there are no a5amino acids) neutralizing activity decreased remarkably composed to No.1with five amino acids. Namely, by the amino acid numerical decrease, theneutralizing activity has a tendency to fall, and particularly No.3,having three amino acids missing in all.

EXAMPLE 4 Effect of a4 and a5 in this Invention on Neutralizing Activity

[0153] To examine the effect of a4 and a5 amino acids on neutralizingactivity, the chain length of No.8 in table 9 were changed. The activitywas measured the same as in example 1. Their results are shown inTABLE12. Face note, NE means “NO EFFECT”. TABLE 12 Anti-HIV neutralizingAmino acid sequence activity (μg/ml) No. a1 a2 A3 a4 a5 HTLV-IIIB KK-1 1Tyr Tyr Lys Ala Ala 78.1 39.1 2 Tyr Tyr Lys Ala — NE 250 3 Tyr Tyr Lys —— NE NE

[0154] From TABLE12, both No.3 with only 3 amino acids(there is not a4and a5 amino acid) and No.2 with only 4 amino acids(there is not a5amino acids) neutralizing activity decreased remarkably compared to No.1with five amino acids. Namely, by amino acid numerical decrease, theneutraizing activity has a tendency to fall, and particularly, No.3having three amino acids missing in all.

EXAMPLE 5 Effect of a4 and a5 in this Invention on Neutralizing Activity

[0155] To examine the effect of a4 and a5 on neutralizing activity, No.1peptide in TABLE 13 was used as a positive control, while we used apeptide(No.2) that kind of a4 amino acids was changed to leucine,hydrophobic amino acid of same as alanine , or a peptide(No.3) that waschanged to proline. Then, we examined the effect to neutralizingactivity as in EXAMPLE 1. The results are shown jointly in TABLE 13.TABLE 13 Anti-HIV neutralizing activity Amino acid sequence (μg/ml) No.a1 a2 a3 a4 a5 HTLV-IIIB KK-1 1 Tyr Tyr Lys Ala Ala 78.1 39.1 2 Tyr TyrLys Leu Leu 31.3 62.5 3 Trr Tyr Lys Pro Pro NE NE

[0156] From TABLE 13, the kinds of a4 and a5 amino acids differed fromthe amino acid that was identified by this invention on No.1, theneutralizing activity decreased or disappeared. The results suggest thatthese kinds of a4 and a5 amino acid have an important effect on theexpression of the activity.

EXAMPLE 6 Effect of A4 and A5 in this Invention on Aglutinin Test

[0157] We examined the effect of A4 and A5 on agglutinin test as shownin TABLE 14. The No.1 peptide was used as a positive control, while weused a peptide (No.2) that kind of A4 amino acids was changed toleucine, hydrophobic amino acid of same as alanine, or a peptide(No.3)that was changed to aspartic acid, acidic amino acid; similarly, thepeptide(No.4) was changed to proline, and the peptide(No.5) was changedto glutamic acid. Then, we examined the effects on neutralizing activitythe same as in EXAMPLE 2. The results are shown jointly in table 14.Face note, ± means “an agglutinin in trace degree”. TABLE 14 Agglutinintest Amino acid sequence Colloidal No. A1 A2 A3 A4 A5 gold conjugatedgp120 1 Asp Val Lys Ala Gly +++ 2 Asp Val Lys Pro Gly − 3 Asp Val LysAsp Gly − 4 Asp Val Lys Ala Pro − 5 Asp Val Lys Ala Glu ±

[0158] From TABLE 14, when a kind of A4 and A5 amino acid on No. 1 wereexchanged to a different amino acid, the agglutinin was negative, ordecreased to a trace degree. These results suggest that the kind of A4and A5 amino acids have important effects on agglutinin test.

EXAMPLE 7 Effect of Macromolecularization on Neutralizing Activity

[0159] We examined the effect of the peptide binding macromolecule onneutralizing activity. An inactive alkaline phosphatase (Alp) was usedas the macromolecule, and it was bound to the peptide that was preparedin SYNTHESIS 6, the same as in EXAMPLE 1. No.8 peptide was used as apositive control on TABLE 3, while unbound inactive alkaline phophatasewas used as negative control. These results are shown jointly in TABLE15. Face note, NE means “negative”. TABLE 15 Anti-HIV neutralizingactivity(μg/ml) HTLV-IIIB KK-1 Macro-molecule Inactive Alp binding125-250 62.5 peptide Positive control peptide 125 125 Negative controlinactive Alp NE NE

[0160] From TABLE 15, when the peptide in this invention bound toprotein already known and grew up to a macromolecule, the solubilityimproved and the neutralization activity increased too.

EXAMPLE 8 The Antibody-like Effect by Doing Induction of the PeptideMore than Once

[0161] After the peptide binding α-cyclodextrin prepared in SYNTHESIS 2in human serum was suspended, we examined whether we could detect with acommercial diagnosis medicine kit for HIV(Dinabot Co., ┌HIV-1/HIV-2EIA┘). The judgment principle of this HIV diagnosis medicine kit wasthat it detects an anti-HIV antibody formed in patient serum, andparticularly, it's good point is that it can detect IgM antibodyappearing at an early stage of HIV infection. The result is shown inFIG. 1.

[0162] As is shown in FIG. 1, the above diagnosis medicine, whichdetects only anti-HIV antibodies present in HIV patient's serum, candetect depending on the content peptide induced binding toα-cyclodextrin. From this result, the peptide in this invention has anaffinity to HIV, and it clearly showed an antibody like effect wheninduction of the peptide was done more than once.

EXAMPLE 9 The Affinity of the Peptide to gp120(1)

[0163] The peptide bound to Sephadex 6MB that was prepared in SYNTHESIS7 was added to the various kinds of density prepared horseradishperoxidase (HRP) labeled HIV-1-gp120(Immuno Diagnosis Co.) and enzymeunlabeled HIV-1-gp120 previously in each and constant disassociation(kd)of peptide in this invention caliculated by drawing up a Schacherd Plotwe calculated was kd=2.14×10⁻¹⁰M.

[0164] From this result, it is clear that the peptide in this inventionhas an affinity of equal to, or greater than the antibody.

EXAMPLE 10 An Affinity of the Peptide to gp120(2)

[0165] The peptide binding Sephadex 4B that was prepared in SYNTHESIS 7was added to the various kinds of density prepared horseradishperoxidase (HRP) and labeled HIV-1-gp120(Immuno Diagnosis Co.) andenzyme unlabeled HIV-1-gp120 previously in each and constantdisassociation(kd) of the peptide in this invention calculated bydrawing up a Schacherd Plot we calculated was kd=4.97×10⁻¹⁰M.

[0166] From this result, it is clear that the peptide in this inventionhas an affinity of equal to, or greater than the antibody.

EXAMPLE 11 A Number of Recognition Site of the Peptide to gp120 (1)

[0167] To confirm the specificity of the peptide in this invention, wemade “peptide conjugated latex beads” that labeled peptide of No.1 inTABLE 1 and No.6 in TABLE 3 on red colored latex beads(Polyscience Co.,200 nm in diameter). When pseudo HIV-1 solution(gp120 conjugatedcolloidal gold) was added in 200 μl of this latex beads solution, redagglutination that could be observed by the naked eye promptly andeasily appeared. When the unlabelled gp120 solution(1 μg protein/ml)that was not conjugated with colloidal gold was added to it, it did notagglutinate at all. For this reason, we inferred that the peptide inthis invention bound only to the location of one site in gp120. Theresults are shown in TABLE 16. TABLE 16 Colloidal gold conjugated gp120gp120 protein Agglutinin test protein contents(1 μg/ml) contents(1μg/ml) No. 1 in Table 1 Presence Absence No. 6 in Table 3 PresenceAbsence

EXAMPLE 12 The Specificity Test

[0168] By using “peptide conjugated latex beads”, latex aggutinationtest medicine of 2 kinds of peptide (No. 1 in Table and No. 6 in Table3) that were prepared with Enforcement example 7, we examined whethernonspecific binding existed. The virus that was in use in thisenforcement example was serum, laboratory strain and a freshly isolatedstrain that was gathered from patients of hepatitis C or B at finalstage. Then, we used virus lysate that conjugated colloidal gold aspreviously. Titer or number (No.) used virus and presence ofagglutination are shown in TABLE 17. TABLE 17 Presence of agglutininTable 1 Table 3 No. Sample/Virus Titer or No. No. 1 No. 6 1 HIV-1 IIIB1.0 × 10⁵ TCID₅₀ + + 2 HIV-1 Lav 1 1.0 × 10⁵ TCID₅₀ + + 3 HIV-1 kk-1 1.0× 10^(4.5) TCID₅₀ + + 4 HIV-2 Lav2 1.0 × 10⁶ TCID₅₀ − − 5 Serum of HCVpatients indistinct − − 6 Serum of HBV patients indistinct − − 7Hepatitis B surface 1.6 × 10⁵ units − − antigen(HbsAg) 8 ValaricellaZoster valirus 9,5 × 10⁵ units − − 9 Argubella Zoster 4.9 × 10⁵ units −− valirus(VZV) 10 HTLV-1 virul lysate 1.1 × 10⁵ units − − 11 humancytomegalovirus 5.5 × 10⁵ units − − (HCMV) 12 Epstein-Barr virus 9.9 ×10⁵ units − − (EB virus)

[0169] From TABLE 17, it is clear that the affinity of peptide in thisinvention to gp120 was specific.

EXAMPLE 13 Removal of HIV from the Serum by an HIV Absorbing Column

[0170] We prepared the removing and absorbing ability of HIV-1 by usingthe peptide/Sepharose 4B in this invention that was prepared inSYSNTHESIS 9. The quantity of the peptide introduced in this inventionthat was conjugated to the above carrier was 5 mg per 1 ml in bedvolume.

[0171] First, after 100 μl of the above absorbed carrier was suspendedin PBS(pH7.2) was added to a test tube, it was treated with an autoclavefor 30 minutes at 121° C., and decompressed naturally and the suppliedsample was prepared. In addition, as a virus model of pseudo HIV-1, weused gp120 conjugated with colloidal gold solution that was preparedusing a protein content of gp120 in 1.5 mg/mL The supplied virussolution, the pseudo HIV solution, was suspended with 100% human serum(96% as final content in serum), as a control the same content of pseudoHIV solution was suspended with PBS(pH 7.2, 0% human serum content). Thesupplied virus solution of 24(2.4 mL) volume was added to a test tubewith the antibody-like peptide of 1 volume(100 μl), and was mixed byshaking in a water bus for 2 hours at 37° C. After the incubation, wetook this test tube out and stood it for 30 minutes at room temperature,then, we collected the unabsorbed pseudovirus sample from thissupernatant fluid. We measured the rate of absorption the sample of 540nm, which was provided in this way; the rate of the 94% serum againstthe absorption of the 0% serum (was calculated as 100% in TABLE 18).These results show in TABLE 18. TABLE 18 Content of serum(%) Rate ofabsorption(%)  0 100 94  74

EXAMPLE 14 Removal of HIV from the Serum through the use of an HIVAbsorbing Column

[0172] By using a carrier of cellulose series that covalented with thepeptide in this invention prepared in SYNTHESIS 7, we examined theabsorption and removal of HIV. At this time, the rate of peptide in thisinvention introduced here was about 5 mg per 1 mL of the bed volume.

[0173] First, 100 μl of the above absorbed carrier suspended inPBS(pH7.2) was added to the test tube, it was treated with an autoclavefor 30 minutes at 121° C., decompressed naturally, and was prepared assupplied sample. Another, HIV-1 virus solution was also used, kk-1strain, this was freshly isolated from domestic AIDS patients by Ohtakeet al.(Kansenshou zasshi(in Japanese), 64;1284-1294,1990) and frozen.After having rapidly defrosted this KK-1 strain (1×10⁵ TCID₅₀), weultra-centrifuged it and prepared the supplied sample that was suspendedpreviously in 100% of inactivated human serum. Next, 1 bed volume then,24 volume of the supplied virus solution were passed through the column.After the addition, we washed down the serum 5 times against the bedvolume to expel the unbound virus that stayed in the column. The viruscontent was measured by using p24 Antigen ELISA Kit (Cellular productsInc.), we calculated S/CO from Cut Off value based on instructionsattached to the kit. The samples measured were: the supplied sample(virus solution), the unbound sample, the washing solution, andextraction that lyzed binding virus from the carrier (fraction ofabsorbed column) by lyzing buffer, attached in the kit. The fractionthat passed without binding to the column included both this unboundsample and the washing solution. These results are shown in TABLE 19.TABLE 19 p24(S/CO) Virus solution(Starting materials) 2,350 Fraction ofcolumn without 2,209 absorption(column passing solution withoutabsorption) Fraction of column absorption 205

[0174] From TABLE 19, we can understand easily that a little less than10% of the supplied virus was absorbed by the column.

[0175] In the above EXAMPLE, we described the reason for the adoptedmeasurement method of p24 content to evaluate the quantity of the virus.

[0176] In this example, we used KK-1 strain, freshly isolated andsuspended in 100% human serum for the reason that it resembles the stateof the virus in an AIDS patient. To evaluate the content correctly isvery difficult. Even if it is usable in the gp120 ELISA Kit in the HIVlaboratory strain, it cannot be used with a freshly isolated virus suchas KK-1. Rather, this is the reason that we used this virus.Accordingly, we can measure p24 content by the EIA method where there isa core protein having a consensus sequence on first structure ormeasuring by the RT-PCR method. However, both methods have the weakpoint that they measure p24 including the debris. As mentioned above,HIV itself is very unstable and will be broken with time, even duringoperation. It is thought the debris exists already, and it willincrease. Therefore, when the debris increases compared with content ofthe absorbed virus, we cannot understand the result, even if we adoptedboth methods. Accordingly, a judgment by an infection experiment isdesirable in order to measure the content of the virus precisely. But,the freshly isolated KK-1 that was used in this example is difficult toinfect compared with a laboratory strain such as HIV-1 IIIB etc. , andparticularly, a virus solution of high density is needed, far exceedingthe content of column absorption in the infection experiment.Considering these problems, it is very difficult to do the experiment.So, for the reason that it is difficult to measure the absorptioncontent if the content is in a dilute virus solution of this degree, wemeasured the p24 content in this example and calculated S/CO bycomparing it with other data.

EXAMPLE 15 The Binding of HIV to Latex Conjugated Peptide

[0177] We filmed the agglutination condition of HIV by No.2virus/No.1peptide-latex beads in TABLE 17. In detail, after theHIV-1LAV1 laboratory strain was added to the above aggulutinin testmedicine, it stood for 6 hours at 4 C. Afterwards, this agglutinatedsolution was put on a support membrane for observation by electronmicroscope, it was negatively stained with uranyl acetate and aphotograph was taken. Referentially, we show this photograph in FIG. 2

[0178] From FIG. 2, we can observe that the HIV virus binds tightly tothe latex beads conjugated HIV-gp120 affinity peptide, those beads bindmutually and agglutinate.

INDUSTRIAL APPLICABILITY

[0179] As described above, the peptide in this invention is; superior instability, an affinity to gp120, is extremely useful as a medicine totreat HIV as it has neutralizing activity equal to previous antibodymolecules, can be used as to diagnose HIV through agglutination and is amedical tool for removing HIV. It has physical stability and isresistant to autoclave treatment, which is not so of antibody molecules.

What is claimed is:
 1. A peptide having affinity to gp120 represented by formula (1): H-A1-A2-A3-A4-A5-R (in the formula, H means hydrogen, A1 is aspartic acid, lysine, valine, glutamic acid, glycine, asparagine, or tyrosine residue, A2 is valine, aspartic acid, tryptophan, lysine, phenylalanine, isoleucine, leucine, or tyrosine residue, A3 is lysine, valine, aspartic acid, arginine, alanine, or tryptophan residue, A4 is alanine, tryptophan, or glycine residue A5 is glycine, alanine, valine, leucine, isoleucine, serine, threonine, methionine, asparagine, glutamine, histidine, lysine, arginine, phenylalanine, tryptophan, proline, or tyrosine residue, R is OH derived from carboxyl group or NH₂ derived from acid amide group).
 2. A peptide having affinity to gp120 represented by formula (2): A1′-A2-A3-A4-A5-R (in the formula, A1′ means aspartic acid, lysine, valine , glutamic acid, glycine, aspragine, or tyrosine residue, or polypeptide residue that an arbitrary amino acid stood in line in N-terminal side from this amino acid A2, A3, A4, A5 and R has the same meaning as the above).
 3. A peptide having affinity to gp120 represented by formula (3): H-A1-A2-A3-A4-AS′-R′ (in the formula, A5′ means: glycine, alanine, valine, leucine, isoleucine, serine, threonine, methionine, asparagine, glutamine, histidine, lysine, arginine, phenylalanine, tryptophan, proline, or tyrosine residue, or polypeptide residue that an arbitrary amino acid stood in line in C-terminal side from this amino acid, H, A1, A2, A3 and A4 is same meaning as the above).
 4. A peptide having affinity to gp120 characterized in having amino acid sequence of A1-A2-A3-A4-A5.
 5. A peptide having affinity to gp120 represented by Formula (4): H-a1-a2-a3-a4-a5-R (In the formula, H means hydrogen, a1 is tyrosine, arginine, phenylalanine, glycine, tryptophan, histidine, or asparatic acid residue, a2 is arginine, tyrosine, tryptophan, alanine, valine, glutamine, histidine, or lysine residue, a3 is lysine, tyrosine, arginine, glutamic acid, methionine, or tryptophan residue, a4 is glycine, alanine, valine, leucine, isoleucine, serine, threonine, methionine, asparagine, glutamine, histidine, lysine, arginine, phenylalanine, or tryptophan residue a5 is glycine, alanine, valine, leucine, isoleucine, serine, threonine, methionine, asparagine, glutamine, histidine, lysine, arginine, phenylalanine, tyrosine, or tryptophan residue, R is OH derived from carboxyl group or NH₂ derived from acid amide group).
 6. A peptide having affinity to gp120 represented by Formula (5): a1′-a2-a3-a4-a5-R. (In the formula, a1′ means tyrosine, arginine, phenylalanine, glycine, tryptophan, histidine, or asparatic acid residue, or polypeptide residue that an arbitrary amino acid stood in line in N-terminal side from this amino acid, a2, a3, a4, a5 and R have the same meaning as above).
 7. A peptide having affinity to gp120 represented by Formula (6): H-a1-a2-a3-a4-a5′ (In the formula, a5′ is glycine, alanine, valine, leucine, isoleucine, serine, threonine, methionine, asparagine, glutamine, histidine, lysine, arginine, phenylalanine, tyrosine, or tryptophan residue, or polypeptide residue that an arbitrary amino acid stood in line in C-terminal side from this amino acid, H, a1, a2, a3, and a4 have the same meaning as above).
 8. A peptide having affinity to gp120 characterized in having amino acid sequence of a1-a2-a3-a4-a5.
 9. A compound which macromolecule compound having a functional group and/or medicine bound to the peptide according to any of claims 1-8, or a pharmaceutically acceptable salt thereof.
 10. A compound according to claim 9 which is both used for absorbing and removing the carrier, or a pharmaceutically acceptable salt thereof.
 11. Virus aggulutinin test medicine using the peptide according to any of claims 1-8 or the kit including this test medicine. 